Preparation, activation and collection of probiotic bacteria
Probiotic bacteria cultures of L. acidophilus (PTCC 4356) and L. rhamnosus (PTCC 1469) were obtained from Iranian Research Organization for Science and Technology (IROST) and inoculated in MRS-broth (QUELAB, Canada) and incubated at 37 °C for 24 h in aerobic conditions. The probiotic bacteria growth in late-log phase was gathered by means of centrifugation (Eppendorf, Centrifuge 5810 R, Germany) at 3000g for 10 min., and afterward it was washed two times in sterilized distilled water prior to applying to the microencapsulation procedure. In free bacteria samples, after centrifugation, 1 ml distilled water added to the tubes (Ghorbani-Choboghlo et al. 2015; Pourjafar et al. 2016).
Preparation of Eudragit S100 nanoparticles
Eu S100 powder was obtained from Evonik Pharma Polymers (Evonik, D-64275, Darmstadt, Germany). To prepare the Eu S100 nanoparticles, SAS (Supercritical Antisolvent Technique) progression was applied and the option of acetone (Scharlau Chemie S.A, Spain) was provided as a solvent for Eu S100 polymer. 4 mg ml−1 of Eu solution was infused into distilled water small quantity as a supercritical fluid that had been held beneath homogenizing force (Wisetise, DAIHAN Scientific Co., Ltd, Korea) at 9000g and at 35 °C for 10 min. Distilled water included 15 mg l−1 Tween 80 (Merk, Hohenbrunn, Germany) as a surfactant. Note that, the acetone solvent was left out through evaporation and particle size and Polydispersity Index (PDI) of Eu S100 was assessed via Laser Particle Size Analyzer device (Brookhaven Instruments Corporation, USA) (Hu et al. 2012; Yoo et al. 2011).
Preparation of chitosan solution
A 0.4 g low-molecular-weight chitosan (Sigma, USA) was blended with 90 ml distilled water and acidified by means of 0.4 ml of glacial acetic acid (Merk, Darmstadt, Germany). Then, the pH was regulated in 5.6–5.8 by adding 1 mol (M) l−1 NaOH. The resulted solution of chitosan was filtered throughout Whatman #4 paper filter and the volume was adjusted to 100 ml before sterilizing at 121 °C for 15 min. Finally, this solution was refrigerated (5 °C) overnight (Abouhussein et al. 2016; Kanmani et al. 2011; Lee et al. 2004; Rodklongtan et al. 2014).
Microencapsulation of probiotic bacteria and, single and double coating of beads
In this research, extrusion technique was carried out in the encapsulation process illustrated formerly via Krasaekoopt et al. (2004) and Pourjafar et al. (2012). A 4% sodium alginate (Sigma, USA) was blended with distilled water and then sterilized at 121 °C for 15 min, and refrigerated overnight. Following day, 10 ml of per bacterial suspension (2 × 1010 colony forming units (CFU/ml) was added in the sodium alginate solution. In the final step, the mixture of the cell suspension and sodium alginate were injected into sterile 0.1 mol l−1 CaCl2 (Merk, Darmstadt, Germany) solution using sterile insulin syringes (0.2 mm) as possible as we could pressure the syringe to force out the solution extremely fast. The droplets turned into gel spheres straight away (the distance between the CaCl2 solution and the needle was 20 cm), and after 60 min, all the beads were gathered and washed through distilled water (Mandal et al. 2006; Mirzaei et al. 2012; Pourjafar et al. 2012).
For the first coating, the beads were submerged in 100 ml of chitosan solution (0.4 g 100 ml−1) with gentle shaking at 1 g for 40 min on a magnetic stirrer (IKA Labortechnik, Model 79219 Staufen, KG, Germany). Following that, the chitosan single coated beads were gathered and washed through distilled water (Kanmani et al. 2011; Krasaekoopt et al. 2004; Liserre et al. 2007).
In the final stage, for the second coating, the beads were immersed in 100 ml Eu S100 nanoparticles solution (4 mg 100 ml−1) and held for 4 h on the shaker (Badhana et al. 2013; Hu et al. 2012; Yoo et al. 2011). The double-coated beads were gathered and washed with distilled water and employed on the same day (see Fig. 1).
Beads characterization
The diameters of 50 haphazardly picked beads (50 uncoated, 50 single coated and 50 double coated beads) were determined by means of an eyepiece micrometer on an optical microscope (Nikon-Model Alphaphot-2 YS2-T. Japan) at a magnification of 10× (magnification factor for each unit of gradient lens was 10.89 at magnification of 10×). The exterior morphology and the interior appearance of beads were examined using optical microscope at the magnifications of 40× and 100×. For examination of interior and exterior appearance, first we produced some beads larger than our normal beads we had produced (at about 1 mm). Then we prepared a cross section of beads by means of a microtome blade (for this purpose, we put beads on a plate and then divided them by microtome blade and by hand, then we applied gram staining on the cut surface) (Ghorbani-Choboghlo et al. 2015; Mirzaei et al. 2011, 2012). Moreover, Scanning Electron Microscope (SEM) method was used to differentiate between surfaces of the beads with or without a nanoparticles coating.
Enumeration of free and microencapsulated probiotic bacteria
Free bacterial counts were determined by adding 1 ml solution containing bacteria in 9 ml phosphate buffer (0.1 mol l−1, pH 7.0) and 1 ml aliquot dilutions were dispensed onto the plates of the MRS-Salicin-agar (MRS agar; QUELAB, Canada and Salicin; Sigma, USA) for L. acidophilus and MRS-Glucose-vancomycin-agar (MRS agar; QUELAB, Canada, Glucose; Merk, Germany and Vancomycin; Sigma, USA) for L. rhamnosus. In MRS-Salicin-agar, Salicin (10 ml solution at 10% w/v) was added in 90 ml of sterilized MRS agar (Mirzaei et al. 2012; Sultana et al. 2000) and in MRS-Glucose-vancomycin-agar, glucose (10 ml solution at 10% w/v) and vancomycin (50 µg ml−1) were added in 90 ml of sterilized MRS agar (Saxelin et al. 2010).
All enumerated plates of L. acidophilus and L. rhamnosus were incubated at 37 °C for 48 h under aerobic condition. The averages were conveyed as colony forming units per ml of the sample (CFU ml−1). To enumerate the encapsulated probiotic bacteria, at first the captured bacteria were released from the beads. For this purpose, 1 g of the double coated beads were suspended in 9 ml of phosphate buffer (0.1 mol l−1, pH 7.0) after shaking for 60 min on a bag mixer (netech–laboratory, Bag Tech®) at room temperature (Pourjafar et al. 2016; Sultana et al. 2000).
Survival of microencapsulated probiotic bacteria following sequential incubation in simulated gastric and intestinal juice (see Fig. 2)
This investigation was anchored in the technique expressed via Sultana et al. (2000), and especially Krasaekoopt et al. (2004) and Mirzaei et al. (2011). Single coated beads (1 g), double coated beads (1 g) and free bacteria suspension (1 ml) were separately placed in a tube including 9 ml of sterilized simulated gastric juice (0.08 mol l−1 HCl, including 0.2 g 100 ml−1 NaCl, pH 1.55) and incubated for 30, 60, 90, and 120 min at 37 °C. Subsequent to incubation, aliquots of 1 g of single coated beads or 1 g of double coated beads or 1 ml of free bacteria suspensions harvested from simulated gastric juice were added to 9 ml of sterilized simulated intestinal juice (0.05 mol l−1 KH2PO4, pH 7.5, with 1 g 100 ml−1 bile salt). Following that, these tubes were incubated for 150 min at 37 °C. After incubation time, beads were dissolved in phosphate buffer solution and cell count was assessed by “pour plate count method”. Also, in free bacteria sample, 1 ml of free bacteria suspension harvested from simulated intestinal juice was used for cell count by “pour plate count method” (see “Enumeration of free and microencapsulated probiotic bacteria”).
Statistical analyses
The entire statistical analyses were carried out by means of SPSS 22 (IBM) software. The conducted tests were duplicate (n = 2). In the enumeration of the bacteria in each replication, plates with the colonies were counted and their internal concentration mean was used to prevent any internal error. Finally, the mean of two replications was calculated to remove external error. The number of the bacteria was reported in terms of the number of the colonies per 1 ml. After confirmation of normality of data by Kolmogrov-Smirnov test further analysis were carried out using Repeated Measures ANOVA test and P < 0.05 regarded to be significant. The graph (Fig. 5) has been constructed using GraphPad Prism version 6 software.
